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Keywords:

  • differentiation;
  • docosahexaenoic acid;
  • hippocampus;
  • n-3 fatty acid deficiency;
  • neurite growth;
  • primary culture

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

Docosahexanoic acid (22:6n-3; DHA) deficiency during development is associated with impairment in learning and memory, suggesting an important role of DHA in neuronal development. Here we provide evidence that DHA promotes neuronal differentiation in rat embryonic hippocampal primary cultures. DHA deficiency in vitro was spontaneously induced by culturing hippocampal cells in chemically defined medium. DHA supplementation improved DHA levels to values observed in freshly isolated hippocampus. We found that DHA supplementation in culture increased the population of neurons with longer neurite length per neuron and with higher number of branches. However, supplementation with arachidonic, oleic or docosapentaenoic acid did not have any effect, indicating specificity of the DHA action on neurite growth. Furthermore, hippocampal cultures obtained from n-3 fatty acid deficient animals contained a lower DHA level and a neuronal population with shorter neurite length per neuron in comparison to those obtained from animals with adequate n-3 fatty acids. DHA supplementation to the deficient group recovered the neurite length to the level similar to n-3 fatty acid adequate cultures. Our data demonstrates that DHA uniquely promotes neurite growth in hippocampal neurons. Inadequate neurite development due to DHA deficiency may contribute to the cognitive impairment associated with n-3 fatty acid deficiency.

Abbreviations used
AA (20 : 4n-6)

arachidonic acid

BSA

bovine serum albumin

CMF-HBSS

calcium magnesium free Hanks balanced salt solution

DAB

3,3′-diaminobenzidine

DIV

days in vitro

DHA (22:6n-3)

docosahexaenoic acid

DPA (22:5n-6)

docosapentaenoic acid

HRP

horseradish peroxidase

HS

horse serum

LNA (18:3n-3)

α-linolenic acid

MAP2

microtubule associated protein 2

MEM

minimum essential medium

OA (18:1n-9)

oleic acid

PBS

phosphate-buffered saline

PUFA

polyunsaturated fatty acid

Docosahexaenoic acid (DHA, 22:6n-3) is a long chain polyunsaturated fatty acid that accumulates in the nervous system reaching up to 15% concentration of the total fatty acids and is constantly maintained through adulthood. DHA can be synthesized from the shorter chain precursor α-linolenic acid (LNA, 18:3n-3), an essential fatty acid that cannot be synthesized de novo by animal tissues and must ultimately be obtained from the diet (Salem et al. 1986).

It is now well recognized that DHA is essential for normal brain development in animals and humans (Salem 1989; Uauy et al. 2001). DHA accumulates in phospholipids mainly during the perinatal period. In the mammalian brain including human brain, the major accumulation occurs during the last period of gestation concomitant with maximal neurogenesis and neuritogenesis (Green and Yavin 1998). It has been shown that n-3 fatty acid deficiency can decrease the DHA content in the brain by 90% (Ward et al. 1996). It has been also demonstrated that n-3 fatty acid deficiency during development induces impairments of hippocampal-dependent learning and memory tasks as seen with the brightness discrimination learning test (Yoshida et al. 1997) and Morris water maze task (Moriguchi et al. 2000). Loss of DHA in brains of n-3 fatty acid deficient animals can be recovered by restituting an n-3 fatty acid adequate diet containing DHA or LNA, the precursor of DHA (Moriguchi et al. 2001). Furthermore, it has been demonstrated that the brain functions impaired by the loss of DHA can be recovered when brain DHA levels return to normal (Moriguchi and Salem 2003).

Although the precise molecular mechanisms of DHA actions in the brain are still unknown, evidences have shown that the hippocampus is compromised in the n-3 fatty acid deficiency. Yoshida et al. (1997) reported that n-3 fatty acid deficiency decreases the density of synaptic vesicles by 30% in the terminals of the CA1 region, as analyzed by electron microscopy, after a learning task in comparison to non-deficient animals. Also, n-3 fatty acid deficiency decreases the soma size in hippocampus (Ahmad et al. 2002a,b), suggesting that DHA has a role in hippocampal development. It has been documented in a few in vitro studies that DHA promotes differentiation of developing photoreceptors (Rotstein et al. 1998) and enhances neurite growth in PC12 cells (Ikemoto et al. 1997). Nevertheless, to properly explain the adverse effects of n-3 fatty acid deficiency during development on hippocampal dependent learning and memory, it is desirable to determine the similar role of DHA in hippocampus.

The formation, guidance, and stabilization of neurites, i.e. axons and dendrites, are the key features of both the developing and adult nervous system, and are critical for synaptic remodeling during memory (Cline 2001; Poirazi and Mel 2001). The specific neuronal morphology of dendrites determines how the synaptic input is going to be processed, and therefore, it determines the response and ultimately the individual physiology and function of the neuron. Generally, longer neurites and higher numbers of dendritic branches bear more synaptic connections (Jan and Jan 2001). In the present work we evaluated the role of DHA in neurite development in hippocampal neurons by determining the changes in neurite length and branching in comparison to other fatty acids. We found that DHA, among other fatty acids tested, uniquely increased the population of hippocampal neurons with longer neurites and higher number of branches, indicating that DHA has a role in promoting neuronal differentiation. Also, cultured hippocampal neurons obtained from n-3 fatty acid deficient rats showed a decreased neurite growth in comparison to hippocampal neurons from n-3 fatty acid adequate rats. Furthermore, the neurite length of n-3 fatty acid deficient neurons was recovered to the level observed in n-3 fatty acid adequate neurons with DHA supplementation in culture. From these data we suggest that DHA may have an important role in hippocampal neuronal differentiation in vivo.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

Trypsin-EDTA, glutamine, minimum essential medium (MEM), heat inactivated horse serum (HS), neurobasal medium and N2 supplements were purchased from Gibco Invitrogen Corporation (Grand Island, NY, USA). Pyruvate, CMF-HBSS, penicillin, streptomycin, poly d-lysine, (+/–) α-tocopherol (vitamin E), Triton X-100, monoclonal antibody against MAP-2ab, immunoglobulin-free and fatty acid free bovine serum albumin (BSA) were from Sigma (St Louis, MO, USA). Docosahexaenoic acid (22:6n-3, DHA), oleic acid (18:1n-9, OA), arachidonic acid (20:4n-6, AA) and eicosanoic (23:0) acid were obtained from Nu-Chek-Prep (Elysian, MN, USA). Docosapentaenoic acid (22:5n-6, DPA) was a generous gift from Omegatech (currently incorporated Martek, Columbia, MD, USA) and urea crystallized by Nu-Chek-Prep. Mowiol 4-88 mounting medium was purchased from Calbiochem (La Jolla, CA, USA). Monoclonal anti-βIII tubulin and polyclonal anti-GFAP were obtained from Promega (Madison, WI, USA). Cy2-, Cy3- and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Jackson Immunoresearch (West Grove, PA, USA). 3,3′-Diaminobenzidine (DAB) substrate was from Roche (Indianapolis, IN, USA) and eight-well slides were from Lab-Tek (Naperville, IL, USA).

Animals and diets

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

All experimental procedures were carried out in accordance with the policies of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) Animal Care and Use Committee. Timed pregnant (250–300 g) Sprague–Dawley rats were received from Charles River Laboratories (Portage, MI, USA). For the experiments with fatty acid supplementation in culture, rats were mated at Charles River, delivered to NIH at 16 days of pregnancy and fed with NIH-31 diet for 2 days before we obtain the E18 fetuses. For experiments with differential diets rats were mated at Charles River, delivered to NIH at 2 days of pregnancy and fed with either one of two diets for 16 days. Both semisynthetic diets based on the AIN-93G formula (Dyets, Inc., Bethlehem, PA, USA; see Reeves et al. 1993), varied only in fat composition. The n-3 fatty acid deficient diet was formulated with safflower oil because of its low content of α-linolenic acid (18:3n-3; LNA). The final content of LNA was 0.1 mole % of total fatty acid. The n-3 fatty acid adequate diet was supplemented with flaxseed oil as a source of LNA to achieve the final LNA composition to be 2.6 mole % of total fatty acid. The fatty acid composition of each diet is shown in Table 1.

Table 1.  Fatty acid composition of diets (mole %)
Fatty acidNIH – 31N-3 FA deficient*N-3 FA adequate*
  1. *Adapted from Murthy et al. 2002. nd = not detected.

  8:0nd1.22.9
 10:0nd4.95.0
 12:0nd44.241.6
 14:01.916.415.9
 16:016.7ndnd
 18:03.37.87.8
 20:00.30.10.1
 22:00.30.10.1
 24:00.20.10.1
Σ Saturates22.683.582.2
 16:12.0ndnd
 18:1 n-919.72.93.2
 18:1 n-71.20.20.2
 20:1 n-12/150.10.10.1
 20:1 n-90.7ndnd
 22:1 n-90.1ndnd
 24:1 n-90.20ndnd
Σ Monounsaturates24.03.23.5
 18:2 n-644.512.212.7
 20:2 n-60.10.1nd
 20:4 n-60.2ndnd
Σ n-6 polyunsaturates44.812.312.7
 18:3 n-34.40.12.6
 20:5 n-31.8ndnd
 22:6 n-32.5ndnd
Σ n-3 polyunsaturates8.70.12.6

Hippocampal primary cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

Pregnant females were killed by cervical dislocation after the exposure to CO2 inhalation. The culture method described by Banker and Cowan (1997) was used with some modifications. Briefly, hippocampi were removed from rat embryonic brain at 18 days of gestation, dissected in calcium magnesium free Hanks balanced salt solution (CMF-HBSS) containing 10 mm HEPES, washed in the same solution and trypsinized (0.25%) for 15 min at 37°C. After three washes with CMF-HBSS the tissue was allowed to settle and resuspended in MEM supplemented with 1 mm glutamine, 1 mm sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin and 10% heat inactivated horse serum (HS; fatty acid composition is shown in Table 2). For the experiments with cells obtained from n-3 fatty acid adequate or deficient animals, 5% heat inactivated HS was used instead of 10% HS. The hippocampal tissue was passed through a fire polished Pasteur pipette and appropriate dilutions were made to seed cells on poly d-lysine eight-well slides (Lab-Tek, Naperville, IL, USA) at a density of 30 000 cells/cm2. After 2 h, culture medium was changed to neurobasal medium plus N2 supplements that contain progesterone, putrescine, transferrin, sodium selenite and insulin (Bottenstein and Sato 1979). The culture medium also contained 1 mm glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin with or without fatty acid. α-Tocopherol (40 µm) was included in all cultures, including the control, to prevent the fatty acid oxidation. The culture medium was not changed for 6 days when the experiment finished. Under these conditions, cultures contained both neuron and glial cells at an approximately 1:1 ratio.

Table 2.  Fatty acid composition of horse serum (mole %± SD)
Fatty acidHorse serum
 14:00.6 ± 0.05
 16:016.6 ± 0.10
 18:015.9 ± 0.08
 20:00.5 ± 0.01
 24:00.3 ± 0.02
Σ Saturates33.9
 16:11.2 ± 0.01
 18:1 n-915.7 ± 0.03
 18:1 n-70.8 ± 0.01
 20:1 n-90.2 ± 0.00
 24:1 n-91.3 ± 0.23
Σ Monounsaturates19.3
 18:2 n-638.6 ± 0.06
 20:2 n-60.2 ± 0.02
 20:3 n-60.3 ± 0.00
 20:4 n-60.5 ± 0.00
Σ n-6 polyunsaturates39.6
 18:3 n-36.4 ± 0.02
 20:5 n-30.3 ± 0.01
Σ n-3 polyunsaturates6.9

Fatty acid supplementation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

Fatty acids such as DHA, OA, AA, or DPA were complexed with fatty acid free BSA in the presence of α-tocopherol before supplementation. An aliquot of DHA, OA, AA or DPA which had been dissolved in methanol under argon atmosphere, aliquoted and stored at − 70°C, was mixed with α-tocopherol under argon atmosphere and darkness to prevent oxidation of the fatty acids and α-tocopherol. The mixture was dried under argon and then complexed with BSA, vortexed and kept on dry ice until it was dissolved with fresh culture medium. Final concentrations of fatty acids, α-tocopherol and BSA in the culture medium were 1.5 µm, 40 µm and 0.01%, respectively.

Fatty acid analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

For fatty acid analysis, cells were grown on poly d-lysine coated 35 mm plates for 6 days, washed twice with 3 mL phosphate-buffered saline (PBS) containing 0.1% fatty acid free BSA and harvested with a rubber policeman with 0.5 mL cold PBS containing 1 mm EDTA at 4°C. The hippocampal lipids were extracted using the method of Bligh and Dyer (1959) in the presence of 23:0 as the internal standard and transmethylated using BF3-methanol (14% w/v) at 100°C for 2 h (Morrison and Smith 1964). After extraction with hexane, the extracts were dried under N2 and redissolved in hexane for gas chromatographic analysis. Fatty acid methyl esters were injected into an HP 5890 gas chromatograph equipped with a flame ionization detector as described previously (Kim and Salem 1990). Fatty acid methyl esters were identified by comparing retention times to known standards and quantified against the internal standard 23:0.

Immunostaining

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

Cells were fixed with 0.4% paraformaldehyde in PBS, pH 7.4 for 30 min. After permeabilization with 0.1% Triton X-100, cells were blocked for 2 h with PBS containing 10% goat serum and 0.5% immunoglobulin free BSA, and then incubated with the primary antibodies against MAP-2ab (mouse monoclonal 1:250) and/or GFAP (rabbit polyclonal 1:1000). After washing with PBS, cells were incubated with the corresponding Cy2, Cy3 or horseradish peroxidase-conjugated secondary antibody (1:50, 1:100, 1:2500, respectively). Horseradish peroxidase (HRP) conjugated secondary antibody was developed using 3,3′-diaminobenzidine (DAB) following the manufacturer's instructions. Slides were mounted in mowiol 4–88.

Neurite measurements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

To study the effect of DHA on the morphology of dendrites, cells were stained with anti-MAP2 antibody to reveal the somatodendritic compartment after 6 days in culture. The reaction of HRP with DAB allowed the tracing of MAP2 positive neurons with a camera Lucida using a 40x oil objective and the Olympus B41 upright microscope from Olympus America (Opelco, Melville, NY, USA). To minimize bias, neurons were blindly traced by covering the name of the slide. Four fields/well were chosen at random and only non-clustered neurons were traced to ensure the precision of the measurements. The neurite length was determined with the NIH image software Image J 1.25 s. The number of neurites was determined by counting directly from the drawings. At least 40 neurons were measured in every case unless specified.

DHA increases the total neurite length of hippocampal neurons

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

A natural deficiency of highly polyunsaturated fatty acids (PUFA) occurs to brain-derived cells when they are transferred from the tissue to the culture in chemically defined medium (Bourre et al. 1983), serving as a convenient in vitro model of n-3 fatty acid deficiency. Using this approach together with in vitro supplementation of DHA, we were able to modulate the levels of DHA in hippocampal neurons in culture to study its action on neurite growth. Hippocampal cells were cultured for 6 days with N2 supplements which is fatty acid free and fatty acid contents were determined at the beginning and the end of the culture (Fig. 1a). In freshly isolated embryonic hippocampus (E18) the DHA content was 13.8 ± 1.5 pmol/µg protein. After 6 days in culture, hippocampal cells decreased the DHA content to 7.8 ± 1.2 pmol/µg protein, representing a reduction of 43%. By supplementing the culture with 1.5 µm DHA from the first day in culture the DHA content was raised to 14.2 ± 0.3 pmol/µg protein, similar to the level of the original tissue. It was also observed that AA was lost more severely during culture. The AA content in the original tissue was 36.2 ± 3.9 pmol/µg protein and after culturing for 6 days in vitro AA decreased by 62% to 13.9 ± 2.5 pmol/µg protein. The AA level was not recovered with DHA supplementation and it remained at 14.9 ± 0.1 pmol/µg protein, similar to the level observed in non-supplemented cultures. By exogenous supplementation in vitro, we were able to modulate the DHA content specifically without affecting the AA content or other polyunsaturated fatty acids (data not shown).

image

Figure 1. (a) Time line of the experimental design. Fatty acid analyses and neurite measurements were performed in hippocampal neurons after 6 days in vitro (DIV) with or without 1.5 µm DHA supplementation in the culture medium. (b) Representative microphotographs of HRP-stained MAP2 neurons after 6 DIV with and without DHA supplementation (bar = 25 µm).

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To evaluate if DHA supplementation in vitro enhances the neurite growth in hippocampal neuronal cells, we examined the neurite length after 6 days in culture. It is well known that after 6 days in vitro hippocampal neurons have a well defined dendritic arborization (Dotti et al. 1988). Neurons were stained with an antibody against MAP2, a specific marker for the somatodendritic compartment (Fig. 1b) and traced with a camera Lucida. The traces of representative control and DHA-treated hippocampal neurons are shown in Fig. 2(a). The total neurite length per neuron was determined by measuring the individual neurite lengths (primary, secondary and tertiary neurites) with the NIH Image Software and summing them per neuron. In control culture without DHA supplementation the major population of neurons showed total neurite lengths per neuron equal to or below 201–300 µm (Fig. 2b). In contrast, cultures treated with DHA showed an increase in the population of neurons with longer total neurite lengths (201–300 µm and higher) and a remarkable decrease in the number of neurons with shorter total neurite lengths (0–100 and 101–200 µm ranges). Moreover, the sum of total neurite lengths in 40 neurons was 8536 µm for control and increased to 11 721 µm after DHA supplementation. The observed difference in the total neurite length per neuron from control and DHA-treated cells was statistically significant when compared as a group with the anova test. After 3 days in vitro no significant differences were detected in growth of dendrites or axons between control and DHA supplemented cultures (data not shown).

image

Figure 2. Effect of DHA on the total neurite length per neuron after culturing with DHA for 6 days. (a) Camera Lucida drawings of representative neurons in control and DHA supplementation conditions. (b) Frequency distribution of the total neurite length per neuron (sum of all the individual neurites per neuron) in 40 neurons. DHA significantly increased the population with longer total neurite length per neuron in hippocampal cells. anova test F1,78 = 16.8, p = 0.0001.

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To determine if the observed increase in total neurite length was due to longer individual neurites or more dendritic branches, or both, these parameters were analyzed separately. All the neurites, primary, secondary and tertiary were evaluated, although tertiary branches were scarce. As shown in Fig. 3(a), most of the neurites in both control and DHA groups are found in a range of 11–20 µm length in comparison to control neurons. Although the difference was not statistically significant, DHA supplemented neurons showed a tendency to have more neurites with longer length (Fig. 3a). DHA supplementation also increased the number of branches per neuron significantly. As can be seen in Fig. 3(b), the population of neurons with more branches increased with DHA, showing the highest frequency in the group of neurons with 13–15 branches/neuron. In contrast, without DHA, the population of neurons with less number of branches was more frequent as the highest frequency was observed in the group of neurons with 4–6 branches.

image

Figure 3. Effect of DHA on the individual neurite length and the number of branches per neuron. (a) Frequency distribution of the individual neurite length from equal number of neurites in control or DHA supplemented condition. Total 373 neurites from approximately 40 neurons were evaluated. (b) Frequency distribution of the number of branches per neuron in 40 neurons. DHA significantly increased the number of branches in hippocampal neurons. anova test F1,78 = 19.1, p = 0.00004.

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DPA, AA and OA did not increase neurite length: Indication of DHA specificity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

In order to determine whether the observed effect of DHA is specific, we evaluated the effect of other fatty acids such as OA (18:1n-9), AA and docosapentaenoic acid (22:5n-6, DPA) on neurite growth. Both OA and AA are abundant in mammalian brain, and DPA is an n-6 PUFA that significantly increases to compensate the loss of DHA in an n-3 fatty acid deficiency (Galli et al. 1971). After 6 days in vitro, cultures supplemented with OA, AA and DPA showed a total neurite length/neuron distribution similar to control hippocampal neurons (Fig. 4). Under the same condition, only DHA treated cells significantly increased the frequency of neurons with higher total neurite length per neuron compared to control cells. Also, the highest frequency group or mode was shifted from 201 to 300 µm group, as seen in control, OA, AA and DPA supplemented cells (Fig. 4a,c,d) to 401–500 µm group in DHA treated cells (Fig. 4b). In addition, only DHA supplementation increased the sum of neurite lengths in 50 neurons to 20 646 µm in comparison to control (16 837 µm). The sum of neurite lengths in 50 neurons from the culture supplemented with OA, AA and DPA was similar to the control value (Fig. 4).

image

Figure 4. Effect of various fatty acids on the total neurite length per neuron. Hippocampal cultures were incubated with or without 1.5 µm DHA, OA, AA or DPA. The total neurite length per neuron in 50 neurons is shown in each histogram. Only DHA significantly increased the population of neurons with longer total neurite length per neuron in comparison to control cells. anova test F4,245 = 5.4, p = 0.0003.

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In vivo DHA reduction decreases neurite growth in hippocampal neurons

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

We induced prenatal n-3 fatty acid deficiency by feeding pregnant rats with a diet almost devoid of n-3 fatty acids (LNA, 0.1%) from the day 2 to day 18 of pregnancy (Fig. 5a). Control animals received an n-3 fatty acid adequate diet containing 2.6% LNA, the precursor for the synthesis of DHA. At E18, the accretion of DHA in hippocampus was only at the level of 3.3% as shown in Table 3. However, feeding with n-3 deficient diet for 16 days during pregnancy successfully decreased the DHA content to 1.9%, representing 42.4% reduction. As expected, DPA increased from 1.9 to 4.1% in the hippocampus by n-3 fatty acid deficiency. Other fatty acids including AA did not show any significant changes due to the deficient diet. To evaluate if the in vivo reduction of DHA levels can modulate the neurite growth, E18 hippocampal cells from n-3 fatty acid deficient or adequate animals were cultured in chemically defined medium for 6 days in vitro without fatty acid supplementation. n-3 fatty acid deficient neurons had significantly shorter total neurite length per neuron in comparison to n-3 fatty acid adequate neurons. As shown in Fig. 5(b), the major population of n-3 fatty acid deficient neurons had total neurite length per neuron in the 201–300 µm range and below. In contrast, the major population of n-3 adequate neurons had total neurite length per neuron in the 201–300 µm range and over. Supplementation of n-3 fatty acid deficient neurons with DHA increased the frequency of neurons with longer neurites in the ranges of 201–300 µm and over as the n-3 fatty acid adequate neurons, reversing the effect of n-3 fatty acid deficiency. The sum of neurite lengths in 40 neurons was 9576 µm in n-3 fatty acid deficient neurons, 11 428 µm in n-3 fatty acid adequate neurons, and 11 602 µm in n-3 fatty acid deficient neurons supplemented with 1.5 µm DHA for 6 days. In agreement with the observed effect of DHA supplementation in culture (Fig. 3), both longer individual neurites (Fig. 5c) and higher number of branches per neuron (Fig. 5d) appeared to contribute to the longer total neurite length observed in adequate and n-3 fatty acid deficient neurons supplemented with DHA. These data indicated that in vivo n-3 fatty acid deficiency, which decreased the DHA level significantly in embryonic hippocampus, had an adverse effect on neurite growth and this effect was reversed by following DHA supplementation in culture.

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Figure 5. Effect of prenatal n-3 fatty acid deficiency and subsequent DHA supplementation on neurite length and number of branches. (a) Time line of the experimental design. E18 hippocampal cultures were obtained from animals after 16 days of differential diets during pregnancy (E2-E18) and then grown in chemically defined medium in the presence or absence of 1.5 µm DHA for 6 days. (b) Frequency distribution of the total neurite length per neuron in 40 neurons obtained from n-3 fatty acid adequate (Ade) or deficient (Def) animals with (Def + DHA) or without DHA supplementation in vitro. The Def group significantly increased the neuronal population with shorter neurite length per neuron in comparison to the Ade group (U = 580, p = 0.034). The Def + DHA group significantly increased the neuronal population with longer neurite length per neuron in comparison to the Def group (U = 492, p = 0.003). (c) Frequency distribution of the individual neurite length in hippocampal cultures obtained from Ade, Def or Def + DHA group. Total 329 neurites from 30 to 40 neurons were considered for each group. The Def group significantly decreased the individual neurite length in comparison to Ade group (U = 48874, p = 0.031). The DHA + Def group showed a tendency to increase the individual neurite length in comparison to Def group (U = 49951, p = 0.087). (d) Frequency distribution of the Number of Branches per neuron in 40 neurons from Ade, Def or Def + DHA group. The Def group significantly increased the neuronal population with less number of branches in comparison to Ade group (U = 594.0, p = 0.047). The DHA + Def group increased the number of neurons with higher number of branches in comparison to Def group (U = 594.5, p = 0.048). The statistical significance was tested using the non-parametric Mann–Whitney U-test.

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Table 3.  Effect of prenatal n-3 fatty acid deficiency on fatty acid composition of E18 hippocampus
 Fatty acid content (mole %± SD)
AdequateDeficient
  • ***

    p < 0.001 by Student's t-test.

 14:01.9 ± 0.71.8 ± 0.6
 16:032.2 ± 1.032.2 ± 0.5
 18:027.1 ± 3.526.5 ± 0.8
 18:1n-99.9 ± 1.110.1 ± 0.2
 18:1n-74.7 ± 0.65.0 ± 0.2
Σ Saturates and mono unsaturates75.875.6
 20:4n-68.8 ± 0.79.0 ± 0.4
 22:4n-62.7 ± 0.22.6 ± 0.1
 22:5n-61.9 ± 0.24.1 ± 0.5***
 22:6n-33.3 ± 0.31.9 ± 0.2***
Σ Polyunsaturates16.717.6

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References

We demonstrated in this study that hippocampal neurons supplemented with DHA had increased total neurite length per neuron. DHA increased both individual neurite length and number of branches, indicating that DHA promoted neurite growth in vitro. DHA action was specific as supplementation with other PUFAs or OA under identical culture conditions did not affect neurite growth. Consistently, cultured hippocampal neurons obtained from n-3 fatty acid deficient rats showed decreased neurite growth in comparison to neurons obtained from n-3 fatty acid adequate rats. Subsequent DHA supplementation of n-3 fatty acid deficient neurons recovered the neurite growth close to the level observed in n-3 fatty acid adequate neurons.

We found that hippocampal cultures obtained from E18 rat fetuses grown in chemically defined medium for 6 days had a 40% reduction in DHA in comparison to freshly isolated hippocampus. This finding is consistent with a previous report using mouse brain cells (Bourre et al. 1983) where they identified this phenomenon as an in vitro essential fatty acid deficiency. They observed that DHA supplementation in the culture stimulated the proliferation of neuronal cells. In our present study we took advantage of these findings and used the hippocampal primary culture in chemically defined medium as an in vitro model of n-3 fatty acid deficiency, which allows us to modulate the content of DHA in hippocampal cells by supplementation. Moreover, our in vitro model offers the advantage that the fatty acid composition in the specific tissue of interest can be conveniently manipulated without the influence or contribution of any other tissues, in comparison to the in vivo model of n-3 fatty acid deficiency. We think that this model can complement the in vivo model to understand the relevance of n-3 fatty acid in specific tissues.

The DHA level in the hippocampal culture was recovered to the initial level by exogenously applying 1.5 µm DHA to the culture media from the seeding time. Although the circulating concentration of DHA as the free fatty acid in rat plasma has been reported to be 10.6 µm, the concentration of free DHA in brain was found to be as low as 1.3 µm (Contreras et al. 2000), indicating that the DHA concentration used in our study is in the physiological range.

Hippocampus is an important brain structure involved in learning and memory functions (Squire and Zola-Morgan 1991). As the n-3 fatty acid deficiency is often associated with impairment in hippocampal dependent learning and memory (See introduction), it is imperative to understand the role of DHA in hippocampal development. Although it has been reported that DHA has positive effects on neurite outgrowth in PC-12 cells, a pheochromocytoma derived cell line capable of differentiating into a sympathetic neuron and retinal development, DHA effect on hippocampal neuronal development has not been explored. We found in this study that DHA supplementation increased the individual neurite length as well as the number of branches per neuron in hippocampal neuronal culture. In PC12 cells, DHA has been shown to enhance the NGF-induced neurite growth (Ikemoto et al. 1997). Also, it has been reported that in developing photoreceptors in vitro, DHA promotes differentiation forming new apical processes by increasing opsin expression as well as improving opsin localization to the apical processes (Rotstein et al. 1998). These in vitro evidences including our results consistently suggest a role of DHA in neuronal differentiation.

An issue to be examined is the molecular mechanisms by which DHA promotes neurite growth in hippocampal neuronal cells. The mechanisms involved in dendrite growth, arborization, and guidance are complex and not completely understood (Jan and Jan 2003). DHA actions at the molecular level are beginning to be unraveled and several possibilities have been raised. For instance, DHA can modulate signal transduction pathways by increasing the phosphatidylserine (PS) content in neuronal membranes, which in turn promotes the activation of Raf-1 and the PI-3 kinase pathways (Kim et al. 2000; Akbar and Kim 2002). It has been shown that Raf-1 and the PI-3 kinase pathways are involved in inducing neurite growth in PC12 and H19-7 hippocampal cell line (Wood et al. 1993; Kuo et al. 1996; Kobayashi et al. 1997; Kita et al. 1998). Also, it has been reported that DHA stimulates phospholipid synthesis, particularly PS and PE, in differentiated PC12 (Ikemoto et al. 1999). As neurite growth requires newly synthesized membrane components, this mechanism may play a role in promoting the neurite growth.

It has been also demonstrated that DHA acts as an endogenous ligand of the nuclear retinoid receptor RXR that functions as a ligand activated transcription factor (de Urquiza et al. 2000). It is well established that RXR signaling is involved in nervous system development (Solomin et al. 1998) and is essential for normal development (Mascrez et al. 1998). Consistently, microarray analyses have shown that a diet enriched with DHA can induce changes in brain and hippocampal expression of 100 and 23 genes, respectively (Kitajka et al. 2002; Puskas et al. 2003). Also, a reduction of hippocampal NGF, a neurotrophic factor identified as a potent regulator of neurite and dendrite development, has been observed in n-3 fatty acid deficiency (Ikemoto et al. 2000) probably as a consequence of the altered gene expression. Since any of these possibilities can contribute to the effect of DHA observed in our study, further studies will be required to establish the exact mechanism of DHA action in neurite growth.

Due to the presence of astroglial cells in the hippocampal cultures we cannot rule out the possibility that DHA stimulates astroglia to secrete growth factors and indirectly stimulate neurite growth of hippocampal neurons. Currently, the exact function of DHA on astroglia is not clear. It has been reported that DHA can be synthesized from the precursor LNA by astroglial cells but not by neurons (Moore et al. 1991; Moore 2001). It has also been shown that DHA is taken up by neuronal cells and retained, in contrast to astroglia which can release it and make it available to be up-taken by neurons (Garcia and Kim 1997; Kim et al. 1999). In this context, it is possible that the neurite extension promoted by DHA shown in this study may be due to the DHA provided to neurons from astroglia.

Our observation that hippocampal cells obtained from n-3 fatty acid deficient embryos develop shorter neurites supports the idea that DHA can promote neurite growth in vivo. Even though the level of DHA accretion in brain is low at E18 (Green et al. 1999), 16 days of n-3 fatty acid deficient diet during the period from E2 to E18 decreased the DHA level by 42% in hippocampus, which was sufficient to negatively modulate the neurite growth in culture. The potential role of DHA in neuronal development in vivo is also supported by the report indicating a decreased neuronal soma size in hippocampus, hypothalamus, and parietal cortex of 21 days old rats after depleting n-3 fatty acid for 3 generations (Ahmed et al. 2002a,b). Thus, impairment in learning and memory observed in animals with n-3 fatty acid deficiency could be related to inadequate neuronal development including suboptimal neurite growth.

In summary, our findings indicate that DHA uniquely promotes the development of hippocampal neurons in vitro by increasing neurite extension and branching. We suggest that DHA deficiencies in vivo may decrease the dendrite length and number of branches, which in turn would affect the number and quality of synaptic connections during development and in adulthood. Similarly, DHA supplementation in vivo as in vitro may promote differentiation in hippocampal neurons.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Animals and diets
  6. Hippocampal primary cultures
  7. Fatty acid supplementation
  8. Fatty acid analysis
  9. Immunostaining
  10. Neurite measurements
  11. Statistical analyses
  12. Results
  13. DHA increases the total neurite length of hippocampal neurons
  14. DPA, AA and OA did not increase neurite length: Indication of DHA specificity
  15. In vivo DHA reduction decreases neurite growth in hippocampal neurons
  16. Discussion
  17. Acknowledgements
  18. References
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